CONTENTS

1. INTRODUCTION…………………………………………………………3
2. MUTATION ………………………………………………………………5
2.1. TYPES OF MUTATION……………………………………………6
2.1.1 Base pair substitution…………………………………………6
2.1.2 Frame shift mutation………………………………………….7
2.1.3 Repeat expansion……………………………………………...7
2.1.4 Changes in chromosomes……………………………………..7

3. REPAIR MECHANISMS…………………………………………………...8

3.1. DNA REPAIR MECHANISM………………………………………...8

3.1.1 Direct repair…………………………………………………….9

3.1.2 Base excision repair…………………………………………..10

3.1.3 Nucleotide excision repair……………………………………11

3.1.4 Mismatch repair……………………………………………….12

3.2. CHROMOSOMAL ABERRATIONS AND REPAIR………………13

3.2.1 Homologous recombination repair……………………………14

3.2.2 Non homologous end joining repair………………………….15

4. GENOTOXICITY TESTING PROCEDURE USED IN REGULATORY

TOXICITY………………………………………………………………17

1
 4.1. Principles of Genotoxicity testing…………………………………17

4.2. Concept of test batteries……………………………………………17

4.3. Criteria for test battery selection……………………………………21

[Link] OF GENOTIXICITY…………………………………………...19

6. PREVENTION OF GENOTOXICITY……………………………………37

7. CONCLUSION…………………………………………………………....39

[Link]………………………………………………………………40

GENOTOXICITY
2
 1. INTRODUCTION

Mutation refers to a change in the DNA structure of a gene. Substances which can
induce mutations are collectively known as mutagens. All chemicals that produce
DNA damage leading to mutation or cancer are described as genotoxic. A
genotoxic agent is a drug or a chemical which causes changes or aberrations or
mutations in the DNA structure and may lead to cancer. They act by changing the
chromosomal structures, forming rings, breaks, joins etc. These can be identified
by the chromosomal aberration test. Any drug which prevents the genotoxic effect
of clastogenic agents are said to be anti- clastogenic or anti- mutagenic [Link]
mutations in somatic cell are not only involved in the carcinogenesis process but
also play a role in the pathogenesis of other chronic degenerative diseases, such as
atherosclerosis and heart diseases, which are the leading causes of death in the
human population1. Micronucleus test and chromosomal aberration test are used
for studying antimutagenic activity of a drug. One of the best ways to minimize the
effect is to identify the antimutagent(substanceswhich suppress or inhibit the
process of mutagenesis by acting directly on the mechanism of cell) or
desmutagens(substances which somehow destroy or inactivate,partially or fully the
mutagens thereby affecting less cell population) in our diets and increasing their
use2.

In genetics, genotoxicity describes the property of chemical agent that damages

the genetic information within acell causing mutation, which may lead to
[Link] is confused with mutagenicity, it is important to note that all
mutagens are genotoxic, but, not all genotoxic substances are mutagenic. The
alteration can have direct or indirect effects on the DNA leading to mutations3. The
permanent hereditary changes can affect either somatic cells of the organism or

3
germ cells to be passed on to the future generations. Cells prevent this type of
mutation by either DNA repair or apoptosis.

The DNA damage can be in the form of single and double breaks, loss of excision
repair, cross-linking, alkalilable sites point mutations and structural and numerical
chromosomal aberrations. The compromised integrity of the genetic material has
been known to cause cancer. Therefore many technique including Ames assay, in
vitro and in vivo toxicology tests, and Comet assay has been developed to assess
the chemical potential to cause DNA damage that may lead to cancer

Anti-mutagen is described as an agent that reduces the apparent yield of

spontaneous and /or induced mutations. Mechanism of anti-mutagenesis have been
classified in to two major processes

1) Desmutagenesis 2)Bioantimutagenesis

Desmutagenesis means factors acts directly on mutagens or inactivate them and

bioantimutagenesis means factors acts on the process of mutagenesis or repair
DNA damages that result in a decrease in the mutation frequency. Antimutagenesis
is considered as one of the most feasible ways for inhibiting the negative effects of
environmental genotoxicants including carcinogens4. Most human carcinogens are
identified by epidemiological studies. These studies are necessarily long term, as
no effect is expected to be observed until decades after the carcinogenic event or
events. However these studies are costly and exposure levels and effects are
difficult to quantify. Few multiple generation mutation assays has been carried out
using rodents5.

1. Dominant lethal
2. Mouse spot test
3. Heritable translocation test
4
These tests must be carried out on a large scale, and tend to be insensitive; in order
to detect a 1% increase (which is a very strong effect) in carcinogenicity in a
human population, one would need to perform an animal study to such a large
scale as to cost over 25 million dollars. Genotoxicity tests can be defined as in
vitro and in vivo tests designed to detect compounds that induce genetic damage by
various mechanisms. These tests enable hazard identification with respect to
damage to DNA and its fixation6.

2. MUTATIONS
Mutation is a permanent change in the DNA sequence that makes up a gene.
Mutations range in size from one DNA base to a whole chromosome change.

Gene mutations occur in two ways: they can be inherited from a parent (hereditary
mutations or gremlin mutations) or acquired during a person’s lifetime and occur
in the DNA of individual cells (acquired or sporadic mutations). These changes can
be caused by environmental factors such as ultraviolet radiation from the sun, or
can occur if a mistake is made as DNA copies itself during cell division. Acquired
mutations in somatic cells cannot be passed on to the next generation, but the
germline mutations can do.

5
2.1. Types of Mutations.

2.1.1 . Base pair (nucleotide pair) substitutions

These are of two types:
(i) Transitions (purine to purine or pyrimidine to pyrimidine)
(ii) Transversions (purine to pyrimidine or pyrimidine to purine).

The consequences of base substitution mutations in protein coding regions of a

gene depend on the substitution and its location. They may be silent, not resulting
in a new amino acid in the protein sequence, eg. GCA or GCG codons in mRNA
both mean arginine [this is often true in the third position of a codon]. A base
substitution could also result in an amino acid substitution; this is referred to as a
missense mutation. For example, CTC in the DNA sense strand [GAG in mRNA]
will specify a glutamate residue in the protein; this is altered to CAC in the DNA
or GUG in the mRNA, resulting in a valine residue in the beta-globin protein chain
causing sickle-cell anemia. Missense mutations may have very serious
consequences, as in the case of sickle-cell anemia, mild consequences as in the
case of hemoglobin C (a different amino acid substitution in position 6 of beta-
globin) or no phenotype as in the case of two known amino acid substitutions at
position 7 of beta-globin. Finally, base substitutions in a protein coding region may
mutate an amino acid codon to a termination codon or vice versa. The former type,
which results in a prematurely shortened protein, is referred to as a nonsense
mutation. The effects of nonsense mutations are variable depending upon how
much of the truncated protein is present and are required for its function.

6
 2.1.2. Frame shift mutations (Insertions, deletions, and
duplications)

These result from the insertion or deletion of one or more (not in multiples of
three) nucleotides in the coding region of a gene. This cause an alteration of the
reading frame: since codons are groups of three nucleotides, there are three
possible reading frames for each gene although only one is used. E.g. mRNA with
sequence AUG CAG AUA AAC GCU GCA UAA amino acid sequence forms the
first reading frame: met gln ile asn ala ala stop. The second reading frame (UGC
AGA UAA) gives: cys arg stop. A mutation of this sort changes all the
amino acids downstream and is very likely to create a nonfunctional product since
it may differ greatly from the normal protein. Further, reading frames other than
the correct one often contain stop codons which will truncate the mutant protein
prematurely.

2.1.3. Repeat expansion

Nucleotide repeats are short DNA sequences that are repeated a number of times in
a row. For example a trinucleotide repeat is made up of 3-base-pair sequences. A
repeat expansion is a mutation that increases the number of times that the short
DNA sequence is repeated. This type of mutation can cause the resulting protein to
function improperly.

2.1.4. Changes in chromosomes

Changes that affect entire chromosomes or segments of chromosomes can cause

problems with growth, development, and function of the body's systems. These
changes can affect many genes along the chromosome and alter the proteins made

7
by those genes. Conditions caused by a change in the number or structure of
chromosomes are known as chromosomal disorders. These changes can occur
during the formation of reproductive cells or in early fetal development. Many
cancer cells also have changes in their chromosome number or structure. These
changes most often occur in somatic cells during a person’s lifetime.

3. REPAIR MECHANISMS

3.1 DNA REPAIR MECHANISMS

One of the endpoints of genotoxicity is gene mutations. Mutagenic chemicals

cause predominantly gene mutations, which are generally not lethal but can form a
major threat to the integrity of chromosomes and viability of cells. Fortunately,
cells are equipped with several DNA repair systems. Depending on the specific
classes of DNA lesions, one or more DNA repair pathways become active7. Four of
the 5 major DNA repair pathways are involved in the repair of DNA lesions
leading to gene mutations: direct repair, base excision repair (BER), nucleotide
excisions repair (NER) and mismatch repair8. The 5th major repair pathway
involved is single/double strand break repair.

8
3.1.1. DIRECT REPAIR

Direct repair acts by removing or reversing the DNA lesions by a single enzyme
reaction in a basically error- free manner and with high substrate specificity. This
mechanism does not require a template, since the damage they restore only occurs
in one base and there is no involvement of incision of the sugar-phosphate
backbone or base excision9. These lesions can occur due to alkylating agents.
Direct repair is carried out by specific enzymes called alkyl guanine-DNA methyl
transferases (AGMT), which remove the alkyl group from the guanine residue of
DNA and transfers it to one of its own cysteine residues10.

3.1.2. BASE EXCISION REPAIR (BER)

9
Base excision repair (BER) is a cellular mechanism that repairs damaged DNA
throughout the cell cycle. This mechanism protects cells from the deleterious
effects of endogenous DNA damage induced by hydrolysis, reactive oxygen
species and other intracellular metabolites, and is also responsible for the removal
of many lesions induced by ionizing radiation and strong alkylating agents. The
main enzymes involved in BER are DNA glycosylases and AP endonucleases. The
DNA glycosylases are involved in excision of the damaged base, where after the
remaining a-basic site is further processed by AP endonucleases. BER is divided
into short-patch repair (where a single nucleotide is replaced) or long patch
repair11.

BER is described as a highly coordinated pathway of consecutive enzymatic

reactions. However, several distinct BER sub-pathways occur, which are
contingent on the type of damage encountered at the onset as well as throughout
the BER process. BER is typically initiated by the series of lesion-specific DNA
glycosylases that remove the damaged base by cleaving the N-glycosidic bond
linking the base to its corresponding deoxyribose, leading to the production of
anAP or abasic site. At least twelve DNA glycosylases have been identified to date,
each acting upon a single or small number of partially overlapping base lesions12.

10
 3.1.3. NUCLEOTIDE EXCISION REPAIR (NER)

Nucleotide excision repair (NER) is a repair pathway that is involved in the

removal of several kinds of DNA lesions which mainly originate from exogenous
sources like UV light or genotoxic chemicals producing bulky adducts and DNA
cross-links13. NER consists of two different subpathways: global genome repair
(GGR) and transcriptioncoupled repair (TCR). These two sub pathways are only
different in the first step of DNA damage recognition. The first pathway (GGR)
eliminates DNA damage present in the genome overall. The DNA recognition is
11
accomplished by a complex of protein factors (XPC-HR23B and XPE). The
second pathway (TCR)removes lesions from active gene.

[Link] REPAIR (MMR)

Mismatch repair (MMR) is a system that recognizes and repairs erroneous

insertions, and mis-incorporation of bases. These can arise during DNA replication
and MMR is a strand-specific repair. During DNA synthesis, the newly
synthesized (daughter) strand may include incorrect bases. Examples of mismatch
bases include base pairs like G/T or A/C. To repair these mismatched base pairs in
the correct manner, it is very important to discriminate between the newly
synthesized (mismatched) strand and the parental strand. The first step in MMR is
12
recognition of the deformity caused by the mismatch. Thereafter, the template and
the non-template strand are determined and the incorrect incorporated base is
excised and replaced with the correct nucleotide. During the repair process not
only the mismatched nucleotide is removed, but a few or up to thousands of bases

of the newly synthesized DNA strand can be removed and replaced 144.

GENOTOXICITY TESTING PROCEDURE USED IN

REGULATORY TOXICITY

4.1 Principle of genotoxicity testing

Chemicals that exert their adverse effect through interaction with the genetic
material (DNA) of cells are called genotoxic21. Most human carcinogens are
genotoxic in nature. The science of genotoxicity mainly concerns that chemicals,
which induce mutations in various experimental models, may conceivably affect
the incidence of heritable mutations in man22. Genotoxicity tests can be defined as
in vitro or in vivo tests designed to detect drugs, which can induce genetic damage
directly or indirectly by various mechanisms of action. Genotoxicity tests enable
hazard identification with respect to DNA damage and its fixation in the form of
gene mutations, large-scale chromosomal damage, recombination and numerical
chromosome changes. Drugs that are positive in these tests that detect such kind of
damage have the potential to be human carcinogens and/or mutagens i.e., may
induce cancer and/ or heritable defects.

4.2The concept of test batteries

Registration of pharmaceuticals requires a comprehensive assessment of their

genotoxic potential. Extensive reviews have shown that many compounds that are

13
mutagenic in the bacterial reverse mutation (Ames) test are rodent carcinogens.
Addition of in vitro mammalian tests increases sensitivity for detection of rodent
carcinogens and broadens the spectrum of genetic events detected, but also
decreases the specificity of prediction (i.e., increases the incidence of positive
results that do not correlate with rodent carcinogenicity) 23. Nevertheless, a battery
approach is still reasonable because no single test is capable of detecting all
genotoxic mechanisms relevant in tumorigenesis.

The general features of a standard test battery are as follows:

i. Assessment of mutagenicity in a bacterial reverse gene mutation test. This test

has been shown to detect relevant genetic changes in majority of genotoxic rodent
and human carcinogens.

ii. Genotoxicity should also be evaluated in mammalian cells in vitro and/or in

vivo.

In vitro and in vivo tests that measure chromosomal aberrations in metaphase cells
can detect a wide spectrum of changes in chromosomal integrity. Breakage of
chromatids or chromosomes can result in micronucleus formation if an acentric
fragment is produced; therefore, assays that detect either chromosomal aberrations
or micronuclei are considered appropriate for detecting clastogens 24. Micronuclei
can also result from lagging of one or more whole chromosome(s) at anaphase and
thus micronucleus tests have the potential to detect some aneuploidy inducers.

Description of the Two Options for the Standard Battery

The following two options for the standard battery are considered equally suitable

Option 1

14
i. A test for gene mutation in bacteria.
ii. A cytogenetic test for chromosomal damage (the in vitro metaphase
chromosome aberration test or in vitro micronucleus test), or an in vitro
mouse lymphoma Tk gene mutation assay.
iii. An in vivo test for genotoxicity, generally a test for chromosomal
damage using rodent hepatopoietic cells, either for micronuclei or for
chromosomal aberration in metaphase cells.

Option 2

i. A test for gene mutation in bacteria

ii. An in vivo assessment of genotoxicity with two different tissues, usually
an assay for micronuclei using rodent hematopoietic cells and a second in
vivo assay.

Modifications to the Test Battery

The following sections describe situations where modification of the standard

test battery might be advisable

(i)Exploratory Clinical Studies

For certain exploratory clinical studies, fewer genotoxicity assays or different

criteria for justification of the maximum dose in vivo might apply (see ICH M3
(R2) guidance).

(ii) Testing Compounds That Are Toxic to Bacteria

In cases where compounds are highly toxic to bacteria (e.g., some antibiotics),
the bacterial reverse mutation (Ames) test should still be carried out, just as
cytotoxic compounds are tested in mammalian cells, because mutagenicity can

15
 occur at lower, less toxic concentrations. In such cases, any one of the in vitro
mammalian cell assays should also be done (i.e., Option 1 should be followed).

(iii) Compounds Bearing Structural Alerts for Genotoxic Activity

Structurally alerting compounds are usually detectable in the standard test

battery since the majority of structural alerts are defined in relation to bacterial
mutagenicity. A few chemical classes are known to be more easily detected in
mammalian cell chromosome damage assays than bacterial mutation assays.
Thus, a negative result in either test battery with a compound that has a
structural alert is usually considered sufficient assurance of a lack of
genotoxicity. However, for compounds bearing certain specific structural
alerts, modification to standard protocols can be appropriate. The choice of
additional test(s) or protocol modification(s) depends on the chemical nature,
the known reactivity, and any metabolism data on the structurally alerting
compound in question25.

(iv)Limitations to the Use of In Vivo Tests

There are compounds for which many in vivo tests (typically in bone marrow,
blood, or liver) do not provide additional useful information. These include
compounds for which data on toxicokinetics or pharmacokinetics indicate that
the compounds are not systemically absorbed and therefore are not available to
the target tissues. Examples of such compounds are some radioimaging agents,
aluminum-based antacids, some compounds given by inhalation, and some
dermally or other topically applied pharmaceuticals. In cases where a
modification of the route of administration does not provide sufficient target
tissue exposure, and no suitable genotoxicity assay is available in the most
16
exposed tissue, it might be appropriate to base the evaluation only on in vitro
testing. In some cases, evaluation of genotoxic effects at the site of contact can
be warranted, although such assays have not yet been widely used26.

4.3Criteria for test battery selection

For setting up of a battery of tests in genotoxicity testing, it has always been

observed that in vitro tests play a major role due to their high sensitivity and
rapidity. Preliminary tests are designed in such a way that it can detect majority
of the genotoxic carcinogens27. One of the most important criteria considered
for in vitro test evaluation is the relative sensitivity of different cell lines and
their genetic diversity. For assay reproducibility, a more comprehensive
protocol with clear understanding of critical processes (pH shift, high
osmolality, and high ionic strength) involved during assay performance are
necessary28. The in vivo test models are generally designed to see the chemical
effects on the route of exposure, duration of treatment, metabolism and target
organ exposure therapeutically relevant to humans.

The situations are,

i) Compounds, which are highly toxic;

Compounds, which are highly toxic to bacteria and interfere with mammalian
test system, should be tested in two in vitro mammalian cell tests using two
different cell types. The end point should include determination of gene
mutation and chromosome damage.

ii) Compounds bearing structural alerts;

17
 The compounds, which produce negative results in the standard genotoxic test
battery but having structural alerts, need to be subjected to further additional
tests with modified protocols.

iii) Compounds, which are not absorbed;

Compounds, which are not systemically absorbed and therefore not available to
the target tissue (bone marrow or liver), should be solely tested in in vitro
assays. These tests should include a bacterial gene mutation and two in vitro
mammalian cell assays using two different cell types with two different end
points.

iv) Evidences of tumour response

The compounds, which are negative in standard genotoxic test battery but
exhibit carcinogenic potential, should be further tested in appropriate models to
evaluate the mechanism of action associated with the carcinogenic activity.
Additional testing can include exogenous metabolic activation or can include
(a) the liver unscheduled DNA synthesis (UDS) test, (b) 32P post labeling (c)
mutation induction in transgenes and (d) molecular characterization of genetic
changes in tumour related genes.

v) Structurally unique chemical classes

On rare occasions, a completely novel compound in a unique chemical class will

be introduced as a pharmaceutical. When such compounds, which are not subjected
to chronic rodent carcinogenicity bioassays, may be tested further, by employing
genotoxicity testing using additional in vitro and/ or in vivo assays

REVIEW OF GENOTOXICITY

18
Poorly soluble particles such as TiO2 , carbon black , and diesel exhaust t
particles have been evaluated for their genotoxicity using both in vitro and
in vivo assays , since inhalation of these compounds by rats at high
concentrations has been found to lead to tumor formation. Two principle modes
of genotoxic action can be considered for particles, referred to as primary and
secondary genotoxicity. Primary genotoxicity is defined as genetic damage elicited
by particles in the absence of pulmonary inflammation, whereas secondary
genotoxicity implies a pathway of genetic damage resulting from the oxidative
DNA attack by reactive oxygen/nitrogen species (ROS/RNS), generated during
particle-elicited inflammation. Conceptually, primary genotoxicity might operate
via various mechanisms, such as the actions of ROS (e.g., as generated from
reactive particle surfaces), or DNA-adduct formation by reactive metabolites of
particle-associated organic compounds (e.g., polycyclic aromatic hydrocarbons).
Currently available literature data, however, merely indicate that the
tumorigenesis of poorly soluble particles involves a mechanism of secondary
genotoxicity. However, further research is urgently required, since (1) causality
between pulmonary inflammation and genotoxicity has not yet been established,
and (2) effects of inflammation on fundamental DNA damage responses that
orchestrate mutagenesis and carcinogenic outcome,that is, cell cycle arrest,
DNA repair, proliferation, and apoptosis, are currently poorly understood.

Genotoxicity and mutagenicity analyses have a significant role in the

identification of hazard effects of therapeutic drugs, cosmetics, agrochemicals,
industrial compounds, food additives, natural toxins and nanomaterials for
regulatory purposes. To evaluate mutagenicity or genotoxicity, different in vitro
and in vivo methodologies exert various genotoxicological endpoints such as point
mutations, changes in number and structure of chromosomes. Areas covered: This

19
review covered the basics of genotoxicity and in vitro/in vivo methods for
determining of genetic damages. The limitations that have arisen as a result of the
common use of these methods were also discussed. Finally, the perspectives of
further prospects on the use of genotoxicity testing and genotoxic mode of action
were emphasized. Expert opinion: The solution of actual and practical problems of
genetic toxicology is inarguably based on the understanding of DNA damage
mechanisms at molecular, subcellular, cellular, organ, system and organism levels.
Current strategies to investigate human health risks should be modified to increase
their performance for more reliable results and also new techniques such as
toxicogenomics, epigenomics and single cell approaches must be integrated into
genetic safety evolutions. The explored new biomarkers by the omic techniques
will provide forceful genotoxicity assessment to reduce the cancer risk. With the
need to understand the potential biological impact of the plethora of nanoparticles
(NPs) being manufactured for a wide range of potential human applications, due to
their inevitable human exposure, research activities in the field of NP toxicology
has grown exponentially over the last decade. Whilst such increased research
efforts have elucidated an increasingly significant knowledge base pertaining to the
potential human health hazard posed by NPs, understanding regarding the
possibility for NPs to elicit genotoxicity is limited. In vivo models are unable to
adequately discriminate between the specific modes of action associated with the
onset of genotoxicity. Additionally, in line with the recent European directives,
there is an inherent need to move away from invasive animal testing strategies.
Thus, in vitro systems are an important tool for expanding our mechanistic insight
into NP genotoxicity. Yet uncertainty remains concerning their validity and
specificity for this purpose due to the unique challenges presented when correlating
NP behaviour in vitro and in vivo. This review therefore highlights the current state
of the art in advanced in vitro systems and their specific advantages and
20
disadvantages from a NP genotoxicity testing perspective. Key indicators will be
given related to how these systems might be used or improved to enhance
understanding of NP genotoxicity.

Food contact materials are all materials and articles intended to come directly or
indirectly into contact with food. Before being included in the positive European
"Union list" of authorized substances (monomers, other starting substances and
additives) for plastic food contact materials, the European Food Safety Authority
(EFSA) must assess their safety "in use". If relevant for risk, the safety of the main
impurities, reaction and degradation products originating from the manufacturing
process is also evaluated. Information on genotoxicity is always required
irrespective of the extent of migration and the resulting human exposure, in view
of the theoretical lack of threshold for genotoxic events. The 2008 EFSA approach,
requiring the testing of food contact materials in three in vitro mutagenicity tests,
though still acceptable, is now superseded by the 2011 EFSA Scientific
Committee's recommendation for only two complementary tests including a
bacterial gene mutation test and an in vitro micronucleus test, to detect two main
genetic endpoints (i.e., gene mutations and chromosome aberrations). Follow-up of
in vitro positive results depends on the type of genetic effect and on the substance's
systemic availability. In this study, we provide an analysis of the data on
genotoxicity testing gathered by EFSA on food contact materials for the period
1992-2015. We also illustrate practical examples of the approaches that EFSA took
when evaluating "non standard" food contact chemicals (e.g., polymeric additives,
oligomer or other reaction mixtures, and nanosubstances). Additionally, EFSA's
experience gained from using non testing methods and/or future possibilities in this
area are discussed. Environ. Mol. Mutagen. 58:361-374, 2017.

21
Genotoxicity and mutagenicity analyses have a significant role in the identification
of hazard effects of therapeutic drugs, cosmetics, agrochemicals, industrial
compounds, food additives, natural toxins and nanomaterials for regulatory
purposes. To evaluate mutagenicity or genotoxicity, different in vitro and in vivo
methodologies exert various genotoxicological endpoints such as point mutations,
changes in number and structure of chromosomes. Areas covered: This review
covered the basics of genotoxicity and in vitro/in vivo methods for determining of
genetic damages. The limitations that have arisen as a result of the common use of
these methods were also discussed. Finally, the perspectives of further prospects on
the use of genotoxicity testing and genotoxic mode of action were emphasized.
Expert opinion: The solution of actual and practical problems of genetic toxicology
is inarguably based on the understanding of DNA damage mechanisms at
molecular, subcellular, cellular, organ, system and organism levels. Current
strategies to investigate human health risks should be modified to increase their
performance for more reliable results and also new techniques such as
toxicogenomics, epigenomics and single cell approaches must be integrated into
genetic safety evolutions. The explored new biomarkers by the omic techniques
will provide forceful genotoxicity assessment to reduce the cancer risk.

With the need to understand the potential biological impact of the plethora of
nanoparticles (NPs) being manufactured for a wide range of potential human
applications, due to their inevitable human exposure, research activities in the field
of NP toxicology has grown exponentially over the last decade. Whilst such
increased research efforts have elucidated an increasingly significant knowledge
base pertaining to the potential human health hazard posed by NPs, understanding
regarding the possibility for NPs to elicit genotoxicity is limited. In vivo models
are unable to adequately discriminate between the specific modes of action

22
associated with the onset of genotoxicity. Additionally, in line with the recent
European directives, there is an inherent need to move away from invasive animal
testing strategies. Thus, in vitro systems are an important tool for expanding our
mechanistic insight into NP genotoxicity. Yet uncertainty remains concerning their
validity and specificity for this purpose due to the unique challenges presented
when correlating NP behaviour in vitro and in vivo. This review therefore
highlights the current state of the art in advanced in vitro systems and their specific
advantages and disadvantages from a NP genotoxicity testing perspective. Key
indicators will be given related to how these systems might be used or improved to
enhance understanding of NP genotoxicity.

Food contact materials are all materials and articles intended to come directly or
indirectly into contact with food. Before being included in the positive European
"Union list" of authorized substances (monomers, other starting substances and
additives) for plastic food contact materials, the European Food Safety Authority
(EFSA) must assess their safety "in use". If relevant for risk, the safety of the
main impurities, reaction and degradation products originating from the
manufacturing process is also evaluated. Information on genotoxicity is always
required irrespective of the extent of migration and the resulting human exposure,
in view of the theoretical lack of threshold for genotoxic events. The 2008 EFSA
approach, requiring the testing of food contact materials in three in vitro
mutagenicity tests, though still acceptable, is now superseded by the 2011 EFSA
Scientific Committee's recommendation for only two complementary tests
including a bacterial gene mutation test and an in vitro micronucleus test, to detect
two main genetic endpoints (i.e., gene mutations and chromosome aberrations).
Follow-up of in vitro positive results depends on the type of genetic effect and on
the substance's systemic availability. In this study, we provide an analysis of the

23
data on genotoxicity testing gathered by EFSA on food contact materials for the
period 1992-2015. We also illustrate practical examples of the approaches that
EFSA took when evaluating "non standard" food contact chemicals (e.g.,
polymeric additives, oligomer or other reaction mixtures, and nanosubstances).
Additionally, EFSA's experience gained from using non testing methods and/or
future possibilities in this area are discussed. Environ. Mol. Genotoxicity testing
relies on the quantitative measurement of adverse effects, such as chromosome
aberrations, micronuclei, and mutations, resulting from primary DNA damage.
Ideally, assays will detect DNA damage and cellular responses with high
sensitivity, reliability, and throughput. Several novel genotoxicity assays may
fulfill these requirements, including the comet assay and the more recently
developed γH2AX assay. Although they are thought to be specific for
genotoxicants, a systematic comparison of the assays has not yet been undertaken.
In the present study, we compare the γH2AX focus assay with the alkaline and
neutral versions of the comet assay, as to their sensitivities and limitations for
detection of genetic damage. We investigated the dose-response relationships of
γH2AX foci and comet tail I Background

Inhalation of benzene at levels below the current exposure limit values leads to
hematotoxicity in occupationally exposed workers.

Objective

We sought to evaluate Diversity Outbred (DO) mice as a tool for exposure

threshold assessment and to identify genetic factors that influence benzene-induced
genotoxicity.

Methods

24
We exposed male DO mice to benzene (0, 1, 10, or 100 ppm; 75 mice/exposure
group) via inhalation for 28 days (6 hr/day for 5 days/week). The study was
repeated using two independent cohorts of 300 animals each. We measured
micronuclei frequency in reticulocytes from peripheral blood and bone marrow and
applied benchmark concentration modeling to estimate exposure thresholds. We
genotyped the mice and performed linkage analysis.

Results

We observed a dose-dependent increase in benzene-induced chromosomal damage

and estimated a benchmark concentration limit of 0.205 ppm benzene using DO
mice. This estimate is an order of magnitude below the value estimated using
B6C3F1 mice. We identified a locus on Chr 10 (31.87 Mb) that contained a pair of
overexpressed sulfotransferases that were inversely correlated with genotoxicity.

The genetically diverse DO mice provided a reproducible response to benzene

exposure. The DO mice display interindividual variation in toxicity response and,
as such, may more accurately reflect the range of response that is observed in
human populations. Studies using DO mice can localize genetic associations with
high precision.

Intensities at various times following treatment with four prototypical

genotoxicants, methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-
nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide (H2O2) and we
tested whether there is a correlation between the endpoints, i.e., alkali-labile sites
and DNA strand breaks on the one hand and the cell's response to DNA double-
strand breaks and blocked replication forks on the other. Induction of γH2AX foci
gave a linear dose response and all agents tested were positive in the assay. The

25
increase in comet tail intensity was also a function of dose; however, mitomycin C
was almost completely ineffective in the comet assay, and the doses needed to
achieve a significant effect were somewhat higher for some treatments in the
comet assay than in the γH2AX foci assay, which was confirmed by threshold
analysis. There was high correlation between tail intensity and γH2AX foci for
MMS and H2O2, less for MNNG, and none for mitomycin C. From this we infer
that the γH2AX foci assay is more reliable, sensitive, and robust than the comet
assay for detecting genotoxicant-induced DNA damage.

Objective

To realize a follow-up genotoxic study to detect whether the chromosome damage

persisted six years after exposure to the oil.

Setting

Fishermen cooperatives in coastal villages.

Participants

Local fishermen who were highly exposed (n = 52) and non-exposed (n = 23) to oil
seven years after the spill.

Measurements

Chromosome damage in circulating lymphocytes.

Results

26
Chromosome damage in exposed individuals persists six years after oil exposure,
with a similar incidence than those previously detected four years before. A
surprising increase in chromosome damage in non-exposed individual was found
six years after Prestige spill vs. those detected two years after the [Link]
sample size and the possibility of some kind of selection bias should be considered.
Genotoxic results cannot be extrapolated to the approximately 300,000 individuals
who participated occasionally in clean-up tasks.

The persistence of chromosome damage detected in exposed individuals six years

after oil exposure seems to indicate that the cells of the bone marrow are affected.
A surprising increase in chromosome damage in non-exposed individuals detected
in the follow-up study suggests an indirect exposition of these individuals to some
oil compounds or to other toxic agents during the last four years. More long-term
studies are needed to confirm the presence of chromosome damage in exposed and
non-exposed fishermen due to the association between increased chromosomal
damage and increased risk of cancer. Understanding and detecting chromosome
damage is important for detecting cancer in its early stages. The present work is the
first follow-up cytogenetic study carried out in lymphocytes to determine
genotoxic damage evolution between two and six years after oil exposure in same
individuals.

Chalcones present several biological activities and sulfonamide chalcone

derivatives have shown important biological applications, including antitumor
activity. In this study, genotoxic, cytotoxic, antigenotoxic, and anticytotoxic
activities of the sulfonamide chalcone N-{4-[3-(4-nitrophenyl)prop-2-
enoyl]phenyl} benzenesulfonamide (CPN) were assessed using the Salmonella
typhimurium reverse mutation test (Ames test) and the mouse bone marrow
micronucleus test. The results showed that CPN caused a small increase in the
27
number of histidine revertant colonies in S. typhimurium strains TA98 and TA100,
but not statistically significant (p > 0.05). The antimutagenicity test showed that
CPN significantly decreased the number of His+ revertants in strain TA98 at all
doses tested (p < 0.05), whereas in strain TA100 this occurred only at doses higher
than 50 μg/plate (p < 0.05). The results of the micronucleus test indicated that CPN
significantly increased the frequency of micronucleated polychromatic
erythrocytes (MNPCE) at 24 h and 48 h, revealing a genotoxic effect of this
compound. Also, a significant decrease in polychromatic/normochromatic
erythrocyte ratio (PCE/NCE) was observed at the higher doses of CPN at 24 h and
48 h (p < 0.05), indicating its cytotoxic action. CPN co-administered with
mitomycin C (MMC) significantly decreased the frequency of MNPCE at almost
all doses tested at 24 h (p < 0.05).

Genotoxic activity, and also presented a small decrease in MNPCE at 48 h (p >

0.05). Additionally, CPN co-administered with MMC significantly increased
PCE/NCE ratio at all doses tested, demonstrating its anticytotoxic effect. In
summary, CPN presented genotoxic, cytotoxic, antigenotoxic, and anticytotoxic
properties. Glibenclamide is an oral hypoglycemic drug commonly prescribed for
the treatment of type 2 diabetes mellitus, whose anti-tumor activity has been
recently described in several human cancer cells. The mutagenic potential of such
an antidiabetic drug and its recombinogenic activity in eukaryotic cells were
evaluated, the latter for the first time. The mutagenic potential of glibenclamide in
therapeutically plasma (0.6 μM) and higher concentrations (10 μM, 100 μM, 240
μM and 480 μM) was assessed by the in vitro mammalian cell micronucleus test in
human lymphocytes. Since the loss of heterozygosity arising from allelic
recombination is an important biologically significant consequence of oxidative
damage, the glibenclamide recombinogenic activity at 1 μM, 10 μM and 100 μM

28
concentrations was evaluated by the in vivohomozygotization assay. Glibenclamide
failed to alter the frequency of micronuclei between 0.6 μM and 480 μM
concentrations and the cytokinesis block proliferation index between 0.6 μM and
240 μM concentrations. On the other hand, glibenclamide changed the cell-
proliferation kinetics when used at 480 μM. In the homozygotization assay, the
homozygotization indices for the analyzed markers were lower than 2.0 and
demonstrated the lack of recombinogenic activity of glibenclamide. Data in the
current study demoMetabolic syndrome is associated with increased risk of
cardiovascular disease, which could be related to oxidative stress. Here, we
investigated the associations between hepatic oxidative stress and vascular function
in pressurized mesenteric arteries from lean and obese Zucker rats at 14, 24 and 37
weeks of age. Obese Zucker rats had more hepatic fat accumulation than their lean
counterparts. Nevertheless, the obese rats had unaltered age-related level of hepatic
oxidatively damaged DNA in terms of formamidopyrimidine DNA glycosylase
(FPG) or human oxoguanine DNA glycosylase (hOGG1) sensitive sites as
measured by the comet assay. There were decreasing levels of oxidatively
damaged DNA with age in the liver of lean rats, which occurred concurrently with
increased expression of Ogg1. The 37 week old lean rats also had higher
expression level of Hmox1 and elevated levels of DNA strand breaks in the liver.
Still, both strain of rats had increased protein level of HMOX-1 in the liver at 37
weeks. The external and lumen diameters of mesenteric arteries increased with age
in obese Zucker rats with no change in media cross-sectional area, indicating 12
outward re-modelling without hypertrophy of the vascular wall. There was
increased maximal response to acetylcholine-mediated endothelium-dependent
vasodilatation in both strains of rats. Collectively, the results indicate that obese
Zucker rats only displayed a modest mesenteric vascular dysfunction, with no
increase in hepatic oxidative stress-generated DNA damage despite substantial
29
hepatic [Link] that glibenclamide, in current experimental conditions, is
devoid of significant genotoxic effects. This fact encourages further investigations
on the use of this antidiabetic agentGiven the positive results of quercetin in in
vitro genotoxicity studies, the in vivo genotoxic properties of this important dietary
flavonoid warrant testing, especially considering possible high intake via widely
available food supplements. Here, this was done by transcriptome analyses of the
most relevant tissues, liver and small intestine, of quercetin supplemented mice.
Quercetin (0.33%) supplemented to a high-fat diet was administered to mice
during 12 weeks. Serum alanine aminotransferase and aspartate aminotransferase
levels revealed no indications for hepatotoxicity. Microarray pathway analysis of
liver and small intestine showed no regulation of genotoxicity related pathways.
Analysis of DNA damage related genes also did not point at genotoxicity.
Furthermore, a published classifier set of transcripts for identifying genotoxic
compounds did not indicate genotoxicity. Only two transcripts of the classifier set
were regulated, but in the opposite direction compared with the genotoxic
compounds 2-acetylaminofluorene (2-AAF) and aflatoxin B1 (AFB1).

Based on the weight of evidence of three different types of analysis, we conclude

that supplementation with quercetin at ~350 mg/kg bw/day for 12 weeks in mice
showed no up-regulation of genotoxicity related pathways in liver and small
intestine as a chemotherapeutic drug.

Kolaviron biflavonoids have demonstrated antihepatotoxic activity in animal

studies. The present study investigated the possible chemopreventive potential of
kolaviron in inhibiting aflatoxin B1 (AFB1) genotoxicity in HepG2 cells.
Kolaviron inhibition of AFB1-induced cytotoxicity by clonogenic assay and

30
genotoxicity by [3H]thymidine incorporation in unscheduled DNA synthesis were
evaluated, including antioxidant potential of kolaviron determined by its reduction
in the intracellular reactive oxygen species level induced by hydrogen peroxide.
Induction of AFB1-detoxicating enzymes such as cytochrome P450 3A4 (3A4) and
glutathione S-transferases (GSTs) A1-1/ A2-2 (alpha) and M1B (mu) was
determined by reverse transcription polymerase chain reaction (RT-PCR) and
northern blotting for the messages and western immunoblot analysis for protein.
Kolaviron significantly (P < 0.01) and dose-dependently inhibited the cytotoxicity
(by 71.6%) and genotoxicity (47.1%) of AFB1 in HepG2 cells. The antioxidant
potential of kolaviron compared favourably with values for the standard
antioxidant trolox C (53.8% at only 4.5 x 10(-2)-fold kolaviron concentration) but
was below that of butylated hydroxyanisole (58.1% at a ninefold kolaviron
concentration). It induced about threefold increases in the messages for 3A4 and
GSTs alpha and mu, including a twofold increase in GSTalpha protein. Kolaviron
may have chemopreventive potential in inhibition of human AFB1 genotoxicity
and possibly hepatocarcinogenesis. The antitumor effect of the partially purified
polysaccharide from Curcuma zedoaria was studied in mice transplanted with
sarcoma 180 cells. The polysaccharide fraction, CZ-1-III, at dose of 6.25 mg/kg/d
showed 50% inhibition in solid tumor growth. When mice were injected with
fractions, CZ-1 and CZ-1-III, at the dose of 100.0 mg/kg, 91.6% and 97.1% of
tumor growth were inhibited, respectively, indicating that the cytotoxic effect of
polysaccharide on sarcoma 180 cells increases upon increasing the amount of
polysaccharide administered. To assess the genotoxicity of CZ-1-III fraction,
several classical toxicological tests were performed. In Ames test, CZ-1-III did not
show any transformation of revertant with or without S-9 metabolic activating
system, indicating the lack of mutagenic effect of the compound. To assess
clastogenic effect, micronucleus and chromosomal aberration assays were
31
performed using Chinese hamster lung (CHL) fibroblast cells. However, up to
259.0 microg/ml concentration of CZ-1-III, neither micronucleus formation nor
chromosomal aberration was induced regardless of the presence of S-9 metabolic
activating system. Inhibition of CZ-1-III on micronucleus formation induced by
mitomycin C was exhibited in a dose-dependent manner, maximally up to 52.0%.
These results strongly suggest that CZ-1-III, the polysaccharide fraction from C.
Zedoaria, decreases tumor size of mouse and prevents chromosomal mutation.
Mitochondrial toxicity was assessed in the brains of developing Erythrocebus patas
monkey fetuses exposed in utero to the nucleoside analogue drug zidovudine (3'-
azido-3'deoxythymidine or AZT). Pregnant E. patas monkeys were given 0 (n = 5),
10 (n = 3), and 40 (n = 3) mg of AZT/day, equivalent to 21 and 86% of the human
daily dose, for the last half (about 10 weeks) of gestation. Mitochondria were
isolated from fetal cerebrum and cerebellum at birth and mitochondrial
morphology was examined in these tissues by transmission electron microscopy
(TEM). Oxidative phosphorylation (OXPHOS) enzyme specific activities were
measured spectrophotometrically. Mitochondrial DNA (mtDNA) integrity and
quantity were determined by Southern blot and slot blot analysis. In the cerebral
mitochondria, reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase
(complex I) specific activity decreased by 25% in monkeys treated with 40 mg of
AZT/day compared with unexposed monkeys (p > or = .05). At the same AZT
dose in the cerebral mitochondria, succinate dehydrogenase (complex II) and
cytochrome c reductase (complex IV)-specific activities showed dose-dependent
increases (p > or = .05), compared with those in controls. In the cerebellum, no
difference was seen in mitochondrial OXPHOS enzyme activities between
unexposed and exposed fetuses. Furthermore, TEM demonstrated no difference in
mitochondrial morphology in frontal cerebrum or cerebellum from unexposed and
exposed fetuses, and all fetuses had similar amounts of mtDNA in both tissues.
32
Cerebral mtDNA degradation was noted in the highest AZT dosage group, whereas
mtDNA from cerebellum was uneffected. Thus, in fetal patas monkeys given a
human equivalent daily dose of AZT during the last half of pregnancy,
mitochondria in the fetal cerebrum appear to sustain moderate damage, while the
fetal cerebellum mitochondria were not effected.

Isolated epithelial cells from porcine urinary bladders were maintained in dividing
long-term monolayer cultures, and were used as a model system for the urinary
bladder in toxicological studies in vitro. To examine the state of differentiation
during the culture period, the culture system was characterised morphologically by
light and transmission electron microscopy and by immune fluorescence labelling
with antibodies against cytokeratins 7,13 and pan. The cultured cells were
identified as urothelial epithelium by their polarised structure, and by their
expression of several uroepithelial specific morphological features, such as
fusiform vesicles, tight junctions and an asymmetric apical cell membrane.
Additionally, the cells were labelled with anti-cytokeratin 7,13 and pan antibodies,
and negatively with anti-vimentin antibodies. The maintenance of suitable culture
conditions was shown by the stable enzyme activities of (gamma-
glutamyltranspeptidase, alkaline phosphatase and acid phosphatase over a culture
period of 4 weeks. A good viability of the cultured cells under the chosen culture
conditions was shown by the presence of low amounts of lactate dehydrogenase (<
of = 5%) in the culture medium. The activities of the chosen marker enzymes for
cell differentiation (gamma-glutamyltranspeptidase), lysosomes (acid phosphatase)
and luminal membranes (alkaline phosphatase) were relatively stable over the
observed culture period. Enzyme activities involved in metabolism of xenobiotics
were determined, to define the ability for metabolism in cultured cells compared
with bladder tissue in situ. Several constitutive phase I and II enzyme activities
33
were found to be stable during the culture period, indicating that the cultured cells
should be able to metabolise xenobiotics in a comparable manner to the urothelium
in vivo. The cytotoxic effects of xenobiotics were investigated and IC50 values
were determined by means of lactate dehydrogenase leakage and inhibition of
neutral red uptake. The induction of sister chromatid exchanges was used as a
parameter for the genotoxic effects of several xenobiotics. This cell culture system
was found to be a very good screening system for the testing of substances that
affect the bladder, especially aromatic amines. OBJECTIVE: To assess the
genotoxic effects of X-ray radiation on human populations. METHODS: The
single cell gel electrophoresis (SCGE) and cytokinesis-blocked micronucleus
(CBMN) test were applied as biological dosimeters to detect DNA damage and
abnormalities in human peripheral lymphocytes of subpopulation exposed to X-ray
radiation. The subjects were divided into four groups: 12 radiation-patients; 13
intervention-radiation-therapy doctors; 32 radiation-diagnostians; 28 controls.
RESULTS: The average comet lengths of the four groups were 128.17 +/- 4.49
microns, 88.09 +/- 5.39 microns, 72.68 +/- 2.57 microns and 32.87 +/- 0.57
microns, respectively. The difference in average comet length between any two
groups was highly significant (P < 0.01). The average micronucleated cell (MNC)
rates (@1000) of the four groups were 12.33 +/- 0.85, 9.75 +/- 1.02, 8.48 +/- 0.66
and 3.18 +/- 0.36, respectively. The difference of MNC rates of Group 1 vs 3, 1 vs
4, 2 vs 4 and 3 vs 4 was highly significant (P < 0.01), and the difference of Group
1 vs 2 was significant (P < 0.05), but there was no difference of MNC rate in
Group 2 vs 3 (P > 0.05). CONCLUSIONS: This study showed that both the comet
assay and the CBMN test could be used to monitor populations exposed to X-ray
radiation, but the comet assay seems to be more sensitive than the CBMN test.

34
The genotoxicity of steviol, a metabolite of stevia extract, was evaluated for its
genotoxic potential using the comet assay. In an in vitro study, steviol at 62.5, 125,
250, and 500 micrograms/ml did not damage the nuclear DNA of TK6 and WTK1
cells in the presence and absence of S9 mix. In vivo studies of steviol were
conducted by two independent organizations. Mice were sacrificed 3 and 24 hr
after one oral administration of steviol at 250, 500, 1000, and 2000 mg/kg. DNA
damage in multiple mouse organs was measured by the comet assay as modified by
us. After oral treatment, stomach, colon, liver, kidney and testis DNA were not
damaged. The in vivo genotoxicity of stevia extract was also evaluated for its
genotoxic potential using the comet assay. Mice were sacrificed 3 and 24 hr after
oral administration of stevia extract at 250, 500, 1000, and 2000 mg/kg. Stomach,
colon and liver DNA were not damaged. As all studies showed negative responses,
stevia extract and steviol are concluded to not have DNA-damaging activity in
cultured cells and mouse organs.
In this study we report the results of cytogenetic tests, namely a search for
chromosome aberrations (CA) and sister chromatid exchanges (SCEs), performed
on human amniotic fluid cells cultured and treated with Cadmium chloride. The
cells from primary cultures were exposed to CdCl2 at 1 microM and 10 microM
for 24 h. At the higher dose, no metaphases were scored and at the lower dose (1
microM) no effects were evident on cell proliferation, and no chromosome
aberrations were found. In the subsequent experiments we used cells from
subcultures exposed to 1 microM and 5 microM CdCl2. At the 5 microM dose was
evident the induction of chromatid breaks, while the frequency of sister chromatid
exchanges shows a small increase, not statistically significant at the dose of 1
microM. In this study we positively demonstrated that amniotic fluid cells grown
in vitro are reliable for testing various mutagenic or teratogenic substances. With
regard to cadmium treatment results, it is evident a clastogenic effect of cadmium
35
chloride but not a significant induction of SCEs. The genotoxic potentiality of the
crude leaf extract of Casearia tomentosa, a medicinal preparation, has been
evaluated in Swiss albino mice. The extract significantly induced the division-
disruptive chromosomal changes in bone marrow cells as well as in primary
spermatocytes; the latter also exhibited marked increase in synaptic disruptions. A
significant decrease in sperm count was noted. The incidence of structural damages
in chromosomes, however, remained within the range of control level frequency.
This herbal preparation, therefore, appears to be primarily spindle-poisoning in its
action, but not clastogenic. The probable mechanism of this differential
genotoxicity is discussed. Lower alkyl acrylate monomers include methyl-, ethyl-,
n-butyl-, and 2-ethylhexyl acrylate. These acrylates are used in the manufacture of
acrylic polymers and copolymers for plastics, food packaging, adhesives, and
cosmetic formulations. Although there is limited potential for human
environmental exposure, occupational exposure can occur via inhalation and
dermal contact. Recently, new genotoxicity data have been generated, along with
in silico and in vitro read-cross analyses, for these acrylates. The availability of
high-throughput screening (HTS) data through the ToxCast™/Tox21 databases
allows for consideration of computational toxicology and organization of these
data according to the ten key characteristics of carcinogens. Therefore, we
conducted a comprehensive review to evaluate the mechanistic, toxicokinetic,
animal, and human data, including HTS data, for characterizing the potential
carcinogenicity, mutagenicity, and genotoxicity of these acrylates. Toxicokinetic
data demonstrate that these acrylates are metabolized rapidly by carboxylesterase
hydrolysis and conjugation with glutathione. HTS data demonstrated an overall
lack of bioactivity in cancer-related pathways. Overall, the genotoxicity and
mutagenicity data support a cytotoxic, non-genotoxic mechanism for these
acrylates. . At high doses, and secondary to chronic site-of-contact irritation and
36
corrosion, rodent forestomach tumors were induced by oral gavage dosing with
ethyl acrylate, and skin tumors were observed following chronic dermal dosing
with 2-ethylhexyl acrylate in C3H/HeJ inbred mice (a strain with deficiencies in
wound healing), but not in the outbred NMRI strain. For both dermal and
forestomach cancers, tumorigenesis is secondary to high doses and long-term
tissue damage, shown to be reversible. With evidence that these chemicals are not
genotoxic, and that they cause forestomach and dermal tumors through chronic
irritation and regenerative proliferation mechanisms, these acrylates are unlikely to
pose a human cancer hazards;

Inosine pranobex (Methisoprinol, ISO, Isoprinosine) is an immuno-modulatory

antiviral drug that has been licensed since 1971 in several countries worldwide. In
humans, the drug is approved for the treatment of viral infections, and it might
also have therapeutic use in animals. The aims of the presented work were to
investigate the genotoxicity of inosine pranobex on BALB/3T3 clone A1 and
HepG2 cell lines and to elucidate its mutagenicity using the Ames test.

Prevention of genotoxicity

Genotoxic effects such as deletions, breaks and/or rearrangements can lead to

cancer if the damage does not immediately lead to cell death. Regions sensitive to
breakage, called fragile sites, may result from genotoxic agents (such as
pesticides). Some chemicals have the ability to induce fragile sites in regions of the
chromosome where oncogenes are present which could lead to carcinogenic
effects. In keeping with this finding, occupational exposure to some mixtures of
pesticides are positively correlated with increased genotoxic damage in the
exposed [Link] damage is not uniform in its severity across

37
populations because individual vary in their ability to activate or detoxify
genotoxic substances, which lead to variability in their incidence of cancer51.

The metabolism of some chemicals results in the production of reactive oxygen

species which is a possible mechanism of genotoxicity. This is seen in the
metabolism of arsenic which produces hydroxyl radicals, which are known to
cause genotoxic effects52. Similarly, Reactive Oxygen species (ROS) have been
implicated in genotoxicity caused by particles and fibres. Genotoxicity of non-
fibrous and fibrous particles is characterized by high production of ROS from
inflammatory cells53.

Flavonoids have been reported to possess a wide range of biochemical and

pharmacological activities, both potentially detrimental and protective. One of the
effects of flavonoids is the ability to modulate the xenobiotic metabolism. Various
studies have indicated that a potential basis for protection is interference with
enzymes such as cytochrome p450 which plays an important role in metabolic
activation of wide range of carcinogens.

Drugs presently being used as anti-mutagenic agent are busulfan, carmustine,

etoposide etc. Plant-derived polyphenolics and other chemicals with antioxidant
properties have been reported to inhibit the expression of genotoxic activity by
pro-oxidant chemicals. In vitro and in vivo studies with ionizing radiation suggest
that hydroquinone (HQ) may have similar protective [Link] protective effect
of HQ may be due to enzyme induction or a direct anti-oxidant effect of HQ
against oxidants commonly present in the diet54.

38
CONCLUSION

Genotoxicity testing of the drugs and their formulation is undergoing the

revolution. Newer methods are getting developed and are assisting the older ones
with their increased sensitivity and other advantages. The current scenario is also
demanding the testing of excipient and formulation, metabolites, impurities and
effects during pregnancy. Newer methods that are developed may be used for
preliminary screening for genotoxicity yet they need development on the basis of
quantization and the other aspects. A genotoxic agent is a drug or a chemical which
causes changes or aberrations or mutations in the DNA structure and may lead to
cancer. They act by changing the chromosomal structures, forming rings, breaks,
joins etc. These can be identified by the chromosomal aberration test. Any drug
which prevents the genotoxic effect of clastogenic agents are said to be anti-
clastogenic or anti- mutagenic agent. DNA repair mechanisms, metabolism of
harmful chemical clastogens and use of anticancer drugs are the major treatments
for genotoxicity. The drugs which are used for treatment of genotoxicity and also
act as anti-cancer agents are alkylating agents, intercalating agents and enzyme
inhibitors. Plant extracts like flavonoids, ellagic acid etc, are found to possess
pharmacological activity and hence being used as anti-mutagenic agents.

39
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Genotoxicity testing: progress and prospects for the next decade.

Turkez H1,2, Arslan ME1, Ozdemir O1

3 .Stephen J. Evans, 1 ,† Martin J. D. Clift, 1 ,† Neenu Singh, 2 Jefferson de Oliveira
Mallia, 1 Michael Burgum, 1John W. Wills, 3 Thomas S. Wilkinson, 4 Gareth J. S.
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associated with advanced in vitro systems towards the study of nanoparticle
(secondary) genotoxicityMutagenesis. 2017 Jan; 32(1): 233–[Link] online
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4. Bolognesi C1,2, Castoldi AF3, Crebelli R4, Barthélémy E3, Maurici D5, Wölfle

D2,6, Volk K3, CSend toFollow-Up Genotoxic Study: Chromosome Damage Two
and Six Years after Exposure to the Prestige Oil Spill
 Kristin Hildur,Cristina Templado,,Jan-Paul Zock,,Jesús Giraldo,
 Published: July 29, 2015
5. [Link] Genotoxic Study:
Chromosome Damage Two and Six Years after Exposure to the Prestige Oil Spill

 Kristin Hildur,Cristina Templado,Jan-Paul Zock,8Jesús Giraldo,Francisco

Pozo-Rodríguez,Alexandra Frances,
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Published: July 29, 2015

6.[Link] Mol Mutagen. 2017

Jun;58(5):361-374. doi: 10.1002/em.22094. Epub 2018

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Oct;13(10):1089-1098. doi: 10.1080/17425255.2017.1375097. Epub 2017 Sep10.

[Link] review of the current and future challenges associated with advanced in
vitro systems towards the study of nanoparticle (secondary) genotoxicityStephen J.
Evans, 1 ,† Martin J. D. Clift, 1 ,† Neenu Singh, 2 Jefferson de Oliveira
Mallia, 1 Michael Burgum, 1John W. Wills, 3 Thomas S. Wilkinson, 4 Gareth J. S.
Jenkins, 1 and Shareen H. Doak 1 ,* Mutagenesis. 2017 Jan; 32(1): 233–
[Link] online 2016 Nov 4. doi: 10.1093/mutage/gew054

[Link] testing approaches for the safety assessment of substances used in

food contact materials prior to their authorization in the European Union.
Bolognesi C1,2, Castoldi AF3, Crebelli R4, Barthélémy E3, Maurici D5, Wölfle
D2,6, Volk K3, Castle L2.

[Link] Mol Mutagen. 201Genotoxicity testing: Comparison of the γH2AX

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41
[Link] Outbred Mice Identify Population-Based Exposure Thresholds and
Genetic Factors that Influence Benzene-Induced Genotoxicity John E.
FrenchDaniel ,L. Morgan,Keith R. Shockley,Gabriel A. Knudsen,Kim G. Shepard.

13. Herman C. PriceFollow-Up Genotoxic Study: Chromosome Damage Two and

Six Years after Exposure to the Prestige Oil

 Cristina Templado,Jan-Paul Zock,Jesús Giraldo,Francisco Pozo-

Rodríguez,Alexandra Frances,Emma Rodriguez-Rodriguez,

Published: July 29, 2015 March 2015[Link]

by:14

14. Genotoxic, Cytotoxic, Antigenotoxic, and Anticytotoxic Effects of

Sulfonamide Chalcone Using the Ames Test and the Mouse Bone Marrow
Micronucleus Test .Carolina Ribeiro e Silva  ,Flávio Fernandes Veloso
Borges ,Aline Bernardes ,Caridad Noda Perez ,Daniela de Melo e Silva ,Lee Chen-
Chen 

Published: September 3, 2015[Link]

[Link] genotoxic signatures in cord blood cells from neonates exposed in

utero to zidovudine or [Link] Vivanti,a Tayebeh S. Soheili,a Wendy
Cuccuini,b,c Sonia Luce,a Laurent Mandelbrot,d,eJerome Lechenadec,e,f Anne-Gael
Cordier,g Elie Azria,h Jean Soulier,b,c,i,j Marina Cavazzana,a,kStéphane Blanche.

[Link] of In Vivo and In Vitro Genotoxicity of Glibenclamide in

Eukaryotic Cells Juliane Rocha de Sant’Anna,Claudinéia Conationi da Silva
Franco,Paulo Cezar de Freitas Mathias,Marialba Avezum Alves de Castro-Prado 

Published: March 24, 2015[Link]

42
[Link] Oxidative Stress, Genotoxicity and Vascular Dysfunction in Lean or
Obese Zucker Rats. Mille Løhr,Janne K. Folkmann,Majid Sheykhzade,Lars J.
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[Link] effects of cadmium chloride on human amniotic fluid cells cultured

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Sulfonamide Chalcone Using the Ames Test and the Mouse Bone Marrow
Micronucleus [Link] Ribeiro e Silva  ,Flávio Fernandes Veloso
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Alexandre Vivanti,a Tayebeh S. Soheili,a Wendy Cuccuini,b,c Sonia Luce,a Laurent

Mandelbrot,d,eJerome Lechenadec,e,f Anne-Gael Cordier,g Elie Azria,h Jean
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Franco,Paulo Cezar de Freitas Mathias,Marialba Avezum Alves de Castro-Prado 
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[Link] and genotoxicity studies of aspartame. Otabe A1, Ohta F1, Takumi

A1, Lynch Author information Ajinomoto Co., Inc., 1-15-1 Kyobashi, Tokyo,
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sissauga,Ontario,2X7,[Link]:[Link]@intertek.com6Biome
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[Link] and Genotoxic Effects of Fluconazole on African Green Monkey

Kidney (Vero) Cell LineRegianne Maciel dos Santos Correa, Tatiane Cristina
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Burbano, and Marcelo de Oliveira Bahia Author information Article
notes Copyright and License information Disclaimer.

First author names, tittle of the work, volume and edition year, page number.

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